1. Field of the Invention
This invention relates generally to the fields of organic chemistry and biological assays. More specifically, the invention relates to optimized oligonucleotides having improved RNase activation characteristics.
2. Description of the Related Technology
Antisense technology is based on the finding that DNA and/or RNA transcription or translation can be modulated using an oligonucleotide which binds to the target nucleic acid. By exploiting the Watson-Crick base pairing, one can design antisense molecules having a very high degree of specificity for the target nucleic acid. Oligonucleotides having only standard (“natural”) bases and backbones must in general contain at least 17 bases in order to bind with sufficient energy to effectively down-regulate gene expression by activating RNase H.
However, even given DNA or RNA of known sequence, it is still difficult to design an optimally effective antisense molecule. This is because nucleic acids are subject to the formation of a variety of secondary and tertiary structures in vivo, and are frequently coiled, supercoiled, folded, and/or obscured by proteins. Some portions of the target sequence are much more susceptible to binding and hybridization by antisense molecules, while other portions of the target sequence are essentially hidden or unavailable. Typically, 20 to 50 oligonucleotides are tested to find one or more active antisense sites per gene.
Standard methods for selecting antisense sites within pre-mRNA or mRNA sequences are insufficient for the rapid, high-throughput application of antisense to large scale target validation programs. Oligonucleotides must be “custom-synthesized” for each target site within each target gene. A standing library of millions or billions of conventional oligo-nucleotides would be required to successfully target each of the approximately 100,000 human genes. An ordered library of millions of antisense oligonucleotides is beyond the chemical, physical, and organizational tools currently available.